(i) Field of the Invention
This invention relates to the use, in a transgenic plant, of at least a portion of a fungus-responsive promoter to induce (i.e., stimulate), in response to a fungus infection of the plant, the expression of a gene or a DNA fragment substantially selectively in cells of the plant around the site of the fungal infection. The use of the fungus-responsive promoter of this invention is especially valuable in transgenic plants for controlling a foreign gene or a DNA fragment that is to be expressed selectively in the cells of the plant which immediately surround the fungal infection site so as to render the plant resistant to the infecting fungus, particularly a plant-pathogenic fungus.
This invention also relates to a first or fungus-responsive chimaeric gene that can be used to transform a plant and that contains a first foreign DNA that:
a) encodes a product which, when expressed in cells of the plant immediately surrounding a fungal infection site, can either i) kill or at least adversely disturb significantly the plant cells immediately surrounding the fungal infection site or ii) kill, disable or repel one or more fungi in the fungal infection site; and PA1 b) is under the control of at least a portion of the fungus-responsive promoter of this invention. PA1 a) the substantial, preferably complete, absence of expression of the first chimaeric gene in all other plant cells; or PA1 b) the substantial absence and preferably the complete absence, by expression of the second chimaeric gene of this invention, of the effects of any expression of the first chimaeric gene in all other plant cells--thereby rendering the plants resistant to fungal infections. PA1 1) the fungus-responsive promoter, which preferably comprises all or promoter-effective portions of a prp1 promoter, especially all or promoter-effective portions of the prp1-1 promoter, particularly promoter-effective portions of the prp1-1 promoter fragment between nucleotides 1 and 696 of SEQ ID no. 1 described in the Sequence Listing, more particularly the promoter fragment between nucleotides 295 and 567 of SEQ ID no. 1 described in the Sequence Listing, and which can direct transcription of a foreign DNA substantially selectively, preferably selectively, in cells of a plant surrounding, preferably immediately surrounding, a site of an infection of the plant by a fungus, particularly a plant-pathogenic fungus such as a Phytophthora (e.g., P. infestans) or a Cladosporium (e.g., Cladosporium fulvum); PA1 2) the first foreign DNA that encodes a first RNA and/or protein or polypeptide which, when produced or overproduced in cells of the plant which surround, preferably immediately surround, the fungal infection site, either a) kills, disables or repels the fungus or b) kills or at least disturbs significantly the metabolism, functioning and/or development of the plant cells surrounding, preferably immediately surrounding, the fungal infection site, so as to limit further spread of the fungus; and PA1 3) suitable 3' transcription termination signals (i.e., 3' end) for expressing the first foreign DNA substantially selectively, preferably selectively, in cells of the plant which surround, preferably immediately surround, the fungal infection site. PA1 1) the second promoter, such as a relatively weak constitutive promoter (e.g., the nos promoter), which can direct transcription of a foreign DNA in at least cells of the plant other than those surrounding, preferably other than those immediately surrounding, the fungal infection site; PA1 2) the second foreign DNA that encodes a second RNA and/or protein or polypeptide which, when produced or overproduced in at least such other cells of the plant, can inhibit or inactivate the first foreign DNA or the first RNA or protein or polypeptide in at least such other cells of the plant; and PA1 3) suitable 3' transcription termination signals for expressing the second foreign DNA in at least such other cells of the plant.
This invention further relates to a cell of a plant, the genome of which is transformed to contain the first chimaeric gene and optionally a second or restorer chimaeric gene; the second chimaeric gene contains a second promoter that controls a second foreign DNA encoding a product which can inhibit or inactivate the first foreign DNA or its encoded product at least in cells of the plant other than those immediately surrounding a fungal infection site, particularly when the first foreign DNA encodes a product that can kill or adversely disturb significantly such other plant cells.
This invention yet further relates to: a) the fungus-resistant transgenic plant, such as a Solanaceae (e.g., tomato or potato) or Brassicaceae (e.g., oilseed rape), which is regenerated from the plant cell of this invention transformed with the first and optionally the second chimaeric genes of this invention, b) fungus-resistant transgenic plants derived from the regenerated transgenic plant and seeds of such plants, and c) plant cell cultures, all of which consist essentially of the transformed plant cells of this invention.
The plants of this invention are characterized by the fungus-responsive expression of the first chimaeric gene of this invention in plant cells surrounding, preferably immediately surrounding, the fungal infection site and either:
(ii) Description of Related Art
The fungi are a very old group of microorganisms. Harmful fungi cause diseases of man, other animals, and especially plants. About 8000 species of fungi can cause plant diseases, and all plants are attacked by some kind of fungi. Some plant-pathogenic fungi can attack many plant species, others attack only one.
In general, fungal plant diseases can be classified into two types: those caused by soilborne fungi and those caused by airborne fungi. Soilborne fungi cause some of the most widespread and serious plant diseases, such as root and stem rot caused by Fusarium spp. and root rot caused by Phytophthora spp.
Since airborne fungi can be spread long distances by wind, they can cause devastating losses, particularly in crops which are grown over large regions. A number of these pathogens have caused widespread epidemics in a variety of crops. Important diseases caused by airborne fungi are stem rust (Puccinia graminis) on wheat, corn smut (Ustilago maydis) on corn, and late blight disease (Phytophthora infestans) on potato and tomato.
Most of these fungal diseases are difficult to combat, and farmers and growers must use a combination of practices, such as sanitary measures, resistant cultivars, and effective fungicides, against such diseases. Hundreds of million of dollars are spent annually for chemical control of plant-pathogenic fungi. As a result, there is today a real need for new, more effective and safe means to control plant-pathogenic fungi.
It is known that plants possess defense mechanisms against fungal diseases. When a plant recognizes a fungal attack, it can respond by inducing several reactions in its cells immediately surrounding the fungal infection site. Resistance mechanisms are activated by the initial infection, so as to limit the spread of the invading fungal pathogen (Ward et al, 1991). The resistance mechanisms include a localized cell death known as a hypersensitive response, the accumulation of phytoalexins, and lignification (De Wit, 1987). The specificity of these responses, which can be very effective in limiting the spread of a fungal infection, depends on the genetic make-up of the host and the pathogen.
Characterization of the genetic components which control cultivar/race specific host/pathogen interactions is a goal of current molecular plant pathology research. Transcriptional activation of defense-related genes is part of the complex defense system which enables plants to deal with contacts with potential pathogens (Collinge and Slusarenko, 1987; Hahlbrock and Scheel, 1989; Bowles, 1990). The identification of cis-acting elements regulating the expression of defense-related genes has been sought in order to elucidate the process by which signal transduction chains connect the initial recognition of a pathogen by a plant host with its induction of defense reactions (Lamb et al, 1989). As found for several other host/pathogen systems (van Loon, 1985; Hahlbrock and Scheel, 1989), infection of potato with the fungus Phytophthora infestans, which is the causal agent of late blight disease, leads to transcriptional activation of genes encoding enzymes of the phenylpropanoid metabolism and PR-proteins (Fritzemeier et al, 1987; Kombrink et al, 1988; Taylor et al, 1990). Transcription of these genes is induced with similar kinetics in compatible and incompatible interactions of different potato cultivars with different Phytophthora infestans races. The nucleotide and deduced amino acid sequences of one of the "pathogenesis related" (or "PR")-protein genes in potato, i.e., prp1-1, which is a member of the large prp1 gene family (with 10-15 very similar copies per haploid genome), shows striking similarity to the corresponding sequences of a gene encoding the HSP26 heat-shock protein in soybean (Taylor et al, 1990). In situ hybridization experiments showed that the PRP1 transcript accumulates around the site of fungal penetration, but the function of this protein in the defense strategy of potato is not yet clear. The homologous soybean HSP26 protein represents a unique member within a group of low molecular weight heat-shocked proteins of plants, missing some characteristic structural features and appearing in an unusually high relative concentration under a broad variety of stress conditions (Czarnecka et al, 1984; Vierling, 1991) but also having no known role in cell metabolism. No sequence similarity has been found between the protein encoded by the prp-1-1 gene and several know PR-proteins from other Solanaceous species (Taylor et al, 1990).
Most plant genes encoding proteins related to pathogen defense, analyzed to date on the level of cis-acting elements, are also activated by several other stress stimuli like mechanical wounding, light and/or elevated concentrations of heavy metals (Oshima et al, 1990; Schmid et al, 1990; Stermer et al, 1990; Douglas et al, 1991; Joos and Hahlbrock, 1992; Becker-Andre et al, 1991).